Delta4-pregene-7alpha, 17alpha, 21-triol-3, 20-dione and esters thereof



llnite States Patent Ser. No. 20,036

3 Claims. (Cl. 260-39747) This application is a division of Serial No.567,478, filed February 24, 1956, now Patent No. 2,960,513.

This invention relates to, and has for its object, the provision of thefollowing steroids: A -pregnene-7a,l7a,- 2l-triol-3,20-dione(7a-hydr0xy-Compound S) and its esters, particularly esters thereof withorganic hydrocarbon carboxylic acids of less than ten carbon atoms, andtothe preparation of M-CompoundS (i.e., A -pregnadiene-l7u,21-diol-3,20-dione) A -pregnene-7a,17a,21-triol-3,20-dione, useful as anintermediate in the preparation of other steroids, is prepared bysubjecting A -pregnene-17a,21-diol-3,20-dione (Compound S) to the actionof enzymes of the microorganism Diplodia natalensis under oxidizingconditions. This oxidation can best be eflected by either including thesteroid in an aerobic culture of the microorganism, or by bringingtogether, in an aqueous medium, the steroid, air and enzymes ofnon-proliferating cells of the microorga- 'nisrn.

In general, the conditions of culturing Diplodia natalensis for thepurposes of this invention are (except for the inclusion of the steroidto be converted) the same as those of culturing various other molds forthe production of antibiotics and/ or riboflavin, i.e., themicroorganism is aerobically grown in contact with (in or on) a suitablefermentation medium. A suitable medium essentially comprises a source ofcarbon and energy. The latter may be a carbohydrate (such as sucrose,molasses, glucose, maltose, starch or dextrin), a fatty acid, a fatand/or the steroid itself. Preferably, however, the medium includes anassirnilable source of carbon and energy in addition to the steroid.

The source of nitrogenous factors may be organic (e.g., soybean meal,corn steep liquor, meat extract and/or distillers solubies) or synthetic(i.e., composed of simple, synthesizable organic or inorganic compoundssuch as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile-air supply should be maintained during fermentation,for example bythe conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. The steroidmay be added to the culture during the incubation period, or included inthe medium prior to sterilization or inoculation. The preferred (but notlimiting) range of concentration of the steroid in the culture is about0.01 to 0.10%. The culture period may vary considerably, the range ofabout 6 to 96 hours being feasible, but not limiting.

The process yields, inter alia, A -pregnene-7a,l7a,21- triol-3,20-dione,a steroid useful not only as an intermediate in the preparation of othersteroids, but also in common with its mono and diester derivatives, as amineralocorticoid (i.e., an agent causing the retention of sodium andexcretion of potassium). Hence, A -pregnene- 7u,l7u,2l-triol-3,20-dioneand esters thereof, particularly esters with organic hydrocarboncarboxylic acids of less than ten carbon atoms (e.g., the lower alkanoicacids as exemplified by acetic, propionic and enanthic acid, thearalkanoic acids as exemplified by oc-toluic and fl-phenylpropionic, andthe aromatic acids as exemplified by ice benzoic and mor p-toluic acid)can be used in lieu of known salt-retaining steroids, and may beadministered parenterally in the treatment of Addisons disease, beingformulated for such administration in the same type of preparations asdesoxycorticosterone acetate, for example, with concentration and/ ordosage based on the activity ofthe particular steroid.

The esters of A -pregnene-7a,l7u,21-triol-3,20-dione are prepared in theusual manner, as by treatment with the desired acid anhydride or acylhalide in an organic solvent (preferably an organic base such aspyridine) to yield either the 2l-mono ester or the 7a,21-diesterdepending on the ratio of acylating agent to steroid present in thereaction mixture. A -pregnene-7a,17a,2l-triol- 3,20-dione can also, ifdesired, be dehydrated in the usual manner, as by treatment with a base(e.g., methanolic potassium hydroxide) to give A -pregnadiene-17a,21-diol-3,20-dione, which can then, if desired, be esterified to the2.1-ester. A -pregnadiene-17a,21-diol- 3,20-dione and its 21-esters arealso useful as mineralocorticoids. Hence, they can be used in lieu ofknown saltretaining steroids and may be administered parenterally in thetreatment of Addisons disease.

The following examples are illustrative of the invention:

' EXAMPLE 1 A -Pregnene-7a,1 7a,21 -Tri0l-3,20-Di0ne (a) Fermentation.-Afermentation medium of the following composition is prepared:

G. Dextrose 10 Corn steep liquor 6 NH4H2PO4 3 CaCO 2.5 Yeast extrac 2.5Soybean oil 2.2

Distilled water to make one liter.

' neopeptone and 1.5 parts agar to parts water) culture of Diplodianatalensis ATCC No. 9055. or derived strains; the parent organism isobtainable, inter alia, from the American Type Culture Collection,Washington, DC.

The flasks are then mechanically shaken for 69 hours at 25 C. on a 280cycle per minute rotary shaker, after which about 9% (v./v.) istransferred to each of 51 flasks containing 50 ml. of the same medium.After 48 hours incubation, a total of 638 mg. of A -pregnene-lh;21-diol-3,20-dione in 25.5 ml. of methanol (to give 0.25 mg. of steroidper ml. in the fermentation vessel) is added. The flasks are thenincubated an additional 24 hours, after which the flasks are harvested,and the contents filtered through a Seitz pad and washed with water givea final volume of filtrate and washings of 2480 ml.

([2) Isolation of A pregnene 7ot,17oc,21 triol 3,20- di0ne.--The culturefiltrate (2480 ml.) is extracted with three 1500 ml. portions ofchloroform and the combined extracts evaporated to dryness in vacuo. Thecrystalline residue (about 438 mg.) is Washed with hexane andrecrystallized from 95% alcohol. mg. of A-pregnene-7a,17a,2l-triol3,20-dione is obtained, which melts at about245-247 C. and which after additional recryssham?? tallization, melts atabout 248 250 C.; M1 +97 c., 0.3 in 95% alcohol); Age, 241 m, (=16,00 Q)x91 5 2.90 3.00, and 3.08

(OH), 5.91,. (20-lreto), 6.10, 6.20 (A -3-keto) Analysis.-Calcd. for 110 (362.45): C, 69.58; H, 8.34. Found: C, 69.76; H, 8.41.

A -pregnene-7a,17a,21-triol-3,2O-dione can be esterified as illustratedby the following example:

EXAMPLE A -Pregnene-7u,1 7 a,21 -Triol-3,20-Dione 7a, 21 -Diacetat e Asolution of mg. of A -pregnene-7a,17 ,21-triol- 3,20-dione in 0.5 ml. ofpyridine and 0.5 ml. of'acetic anhydride is allowed to stand overnightat room temperature. Removal of the excess reagents in vacuo leaves acrystalline residue (about 26 mg.) which after two recrystallizationsfrom acetone-hexane furnishes the pure A-pregnene-7a,17a,21-triol-3,20-dione diacetate having the followingproperties: MP. about 180182 C; +39 c., 0.40 in CHCl A313 238 m l(e=15,500); $31? 2.88 1 (OH); 575 5.79;; (aoetyl and ZO-keto),6.00M,'6.18/-L (A -3-keto) Analysis.-Calcd. for CH3407 (446.52) C,67.24; H, 7.68. Found: C, 67.63; H, 7.70.

Similarly by substituting other acid anhydrides, such as propionicanhydride, or acyl halides, such as benzoyl chloride, for the aceticanhydride in the procedure of Example 2, the corresponding esterderivatives are produced. Furthermore, if only one mole equivalent ofacetic anhydride is used in the procedure of'Example 2, the21-monoacetate is obtained.

4- Ai-pregnene-h,17a,21 triol 3,20-dione can be .dehy drated asillustrated by the following example:

EXAMPLE 3 AQ- Pr egnadiena-l 7 ;,21 -D fol-3,20-Dgi 0ne A solution ofmg. of A pregnene-7u,17a,21-triol- 3,20-dione-in 2 ml. of 2.5%methanolic potassium hydroxide is allowed to stand at room temperaturefor 24 hours. During this period of time, the ultraviolet maximum at 241mu gradually decreases'and gives way to a maximum at 285'mcharacteristic of the A -3-ketone grouping. 'The' solutio n isneutralized with dilute acetic acid, water is added and the methanol isevaporated in vacuo. .The resulting suspension is extracted withchloroform, the chloroform removed in vacuo and the resulting residuecrystallized from acetone-hexane to give pure A-pregnadiene-17a,21-diol-3,20-dione.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A -pregnene-7a,1 7u,2l-triol-3,20-dione.

2. A pregnene 7a,17a,21 triol 3,20 dione ;,21- diacetate.

3. A compound selected from the group consisting of I the7oc-monoesters, 21-monoesters and 7a,2l-diesters of A-pregnene-7a,17a,21-tri01-3,2O-dione, wherein the ester is derived froman organic hydrocarbon carboxylic acid of less than ten carbon atoms.

Fried et al. July 3, 1956 Shull et al. Feb. 26, 1957

1. $4-PREGNENE-7A,17A,21-TRIOL-3,20-DIONE.